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Biomedical subjects

G Schapira

Publications and source records attributed to G Schapira.

At least 19 recordsLinked to original sources
PubMed1987

Detection of Duchenne muscular dystrophy carriers: quantitative echography and creatine kinasemia.

Data obtained from simultaneous determinations of serum creatine-kinase levels and estimation of ultrasound attenuation values in muscles greatly improved the detection of obligate carriers of Duchenne muscular dystrophy than when only one of these methods was employed alone. Eleven carriers out of 19 had a high creatine-kinasemia level and nine carriers out of 19 had a high (abnormal) attenuation value. Because of the limited overlapping between the two parameters studied, we were able to recognize 17 obligate carriers out of the 19. This indicates that the parameters studied concern different features of the disease, and the practical and theoretical considerations are discussed. The techniques are discussed together with molecular genetic investigations.

Adult↗
PubMed1986

[Female transmitters of Duchenne's myopathy: detection by quantitative ultrasonography combined with blood creatine kinase].

Duchenne muscular dystrophy (DMD) is a disease inherited in an X-linked recessive manner. Nothing is yet known about the gene defect involved. The problem of DMD carrier detection is very important and now without satisfactory solution. Presently the best method, for this detection, is the determination of the serum creatine-kinase (CK) level. However about 30% of DMD children's mothers have normal CK levels. In this study we associated CK determination and a new echographic method. This physical investigation is based on the muscle attenuation measurements through the evolution of the running spectral moments. The parameter expressed is the slope of the ultrasound attenuation signal (in decibels by centimetre and by megahertz; dB . cm-1 . MHz-1). We measured this parameter in vivo on the vastus medialis. The use of the chemical technique and of the physical one improves largely the detection of obligate carriers of Duchenne muscular dystrophy than when one of these methods is employed alone. One such combination recognizes 17 out of the 19 obligate carriers in our series. The difference of the results obtained by these two unrelated methods cannot be yet interpreted, but can reflect genetic heterogeneity in DMD and the inactivation of the X chromosomes.

Creatine Kinase↗
PubMed1985

Detection of tyrosine-specific protein kinases with gastrin as exogenous substrate.

Gastrin was recently shown to be phosphorylated on its single tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine protein kinase (TPK). The TPK previously detected in the murine lymphoma (LSTRA) induced by the Moloney murine leukemia virus phosphorylates gastrin, the apparent Km is 65 microM and the maximum rate 1900 pmol/min per mg; the kinase is more efficient with MnCl2 than with MgCl2, is stimulated by NaVO3 and inhibited by ZnCl2. Gastrin phosphorylation is observed only when a TPK is expressed by the cell: extracts of fibroblasts infected with a temperature-sensitive mutant of the Rous sarcoma virus had no gastrin kinase activity when grown at the non-permissive temperature whereas cells grown at the permissive temperature were transformed and disclosed a clear gastrin kinase activity. Gastrin kinases were detected in various transformed cells: human lymphomas, K562 cells, cells from a patient with acute proliferative leukemia, and normal cells: human T and B lymphocytes.

Animals↗
PubMed1984

A spin label study of the erythrocyte membrane in mothers and sisters of patients suffering from Duchenne muscular dystrophy.

The erythrocyte membranes of mothers and sisters of boys suffering from Duchenne Muscular Dystrophy (DMD) have been studied by spin labelling. Two oxazolidine nitroxide derivatives of stearic acid were used. With the first of them (16 NS) which probes the hydrophobic part of the phospholipids, we measured the fluidity of the membrane as a function of temperature. The second nitroxide derivative (5 NS) probes the membrane near the phospholipid polar heads. The amplitude of the electron spin resonance signal was studied as a function of the spectrometer microwave power in order to determine the paramagnetic label saturation behaviour. No significant difference was observed between the control adult women and the carrier mothers. On the contrary, almost all the normal young premenarchial girls showed simultaneously a break in the fluidity vs. temperature plot of the 16 NS probe and a saturation phenomenon of the 5 NS label signal. In about 50% of the DMD boys' sisters, no break in the temperature plot nor saturation behaviour was observed. This corresponds to the theoretical repartition between normal and carrier girls if one admits that about 30% of the latter do not have any detectable membrane abnormality, as in the case of the creatine kinase (CK) test which shows about 30% of normal levels in carrier women. The study of the erythrocyte membrane in young girls can then be an useful complementary tool to detect DMD carriers.

Adolescent↗
PubMed1984

Solubilization of plasma membranes in anionic, non-ionic and zwitterionic surfactants for iso-dalt analysis: a critical evaluation.

A critical study has been made of the solubilizing properties of anionic, nonionic and zwitterionic surfactants to be used in the first dimension of two-dimensional isoelectric focusing-sodium dodecyl sulphate (IF-SDS) maps. Excess of SDS is a powerful solubilizing agent, but prevents proteins from entering the IF gel. Nonidet P-40 (NP-40)-urea mixtures are mediocre solubilizing agents, but are compatible with the IF dimension. Zwitterionic detergents (zwittergents) appear to exhibit a well balanced solubilizing power and are able to generate sharp two-dimensional maps, with round spots and minimal vertical and horizontal streaking. SB3-12 and SB3-14 appear to have the best solubilizing properties; shorter surfactants (SB3-8) exhibit a poor solubilization efficiency, while longer detergents (SB3-16) bind too strongly to hydrophobic regions in proteins. The random performance of non-ionic detergents has been attributed to their ability to form mixed micelles with the carrier ampholytes used in the IF step: depending on the relative ratio of NP-40 to Ampholines, different types of micelles would be formed, which, when reaching appropriate stoichiometries and charge densities, would mimic the behaviour of natural zwittergents. Acceptable two-dimensional maps can be obtained when the sample is lysed in limiting amounts of SDS (SDS:protein = 1:3), suggesting that excess of free SDS in solution is deleterious to the IF process.

Animals↗
PubMed1984

Identification of a 58,000 daltons phosphoprotein with tyrosine protein kinase activity in a murine lymphoma cell line.

A very high level of tyrosine protein kinase (TPK) activity has been recently detected in a murine lymphoma, induced by Moloney murine leukemia virus. A major endogenous substrate for tyrosine phosphorylation in vitro is a protein of Mr 55-60,000 (p58) associated with the detergent insoluble matrix of LSTRA cells; in the present work p58 was solubilized, isolated by anion exchange chromatography and then precipitated by antiphosphotyrosine antibodies. Through these steps of isolation, TPK activity was measured by the use of a simplified gastrin phosphorylation assay. It is demonstrated that the TPK activity copurifies with p58, which leads to the conclusion that p58 bears itself the enzymatic activity. Although functionally similar to other enzymes of this group, this newly characterized TPK does not seem to be closely related to one of the previously documented TPK. This suggests either that this protein is the product of a so far unrecognized cellular TPK gene or that it derives from a rearrangement of one of the previously described TPK genes.

Animals↗
PubMed1984

The rate of initiation of alpha- and beta-globin mRNA translation is modulated by 50 kDa, 28-kDa and 24-kDa polypeptide-containing fractions.

The relative rates of initiation of alpha- and beta-globin mRNA translation in a Krebs II ascites cell-free system are differently modulated by a 50-kDa protein and two fractions containing either a 28-kDa or a 24-kDa polypeptide. Each of these fractions stimulated a discrete step that limits initiation of protein synthesis, but other rate-limiting steps take place upstream and/or downstream, resulting in characteristic kinetics of the stimulation of alpha- and beta-globin synthesis. The ascites extracts appear to be deficient in these activities.

Animals↗
PubMed1983

Alterations in glycosylation of plasma membrane proteins during myogenesis.

Highly purified plasma membranes were obtained from cells of the L6 line at three characteristic stages of myogenesis: Actively proliferating cells; post-mitotic, confluent myoblasts which have already aligned; and fused myotubes. Differential glycosylation of the plasma membrane proteins of these cells was detected by staining polyacrylamide gels of the separated components with three lectins of different specificity: Concanavalin A (conA), wheat germ agglutinin (WGA) and phytohemagglutinin (PHA) Els. Four kinds of developmentally regulated changes could be identified. 1. Those which took place only at confluency (160, 150, 90, 85, 60, 43 and 40 kD for conA binding, 190 kD for WGA binding, 190 and 110 kD for PHA Els binding. 2. Those which took place only at fusion (135, 51.5 and 38 kD for conA, 160 and 150 kD for WGA and 150 kD for PHA Els binding). 3. Those where the phenomena initiated at confluency continue during fusion (66.5 and 32 kD for conA and 120 kD for PHA binding). 4. Those where opposite changes take place at confluency and at fusion (48 kD for conA, 180, 98 and 85 kD for PHA binding). These results suggest that most developmentally regulated changes in glycosylation take place during the first cell-cell contact step of myogenesis. Metabolic labelling experiments showed that, on the contrary, only few alterations in the accumulation of plasma membrane proteins take place prior to the main burst of fusion.

Animals↗
PubMed1983

Preparation and characterization of mRNAs from rat heart muscle.

RNA was prepared from rat heart muscle by a procedure using guanidium thiocyanate and centrifugation on a CsCl cushion. Analysis of the RNA by sucrose gradient centrifugation and electrophoresis shows that major part of it was long sized. Poly (A+) RNA was isolated with a yield of 35 micrograms/g. The average size of the poly (A) tail was 120 nucleotides. RNA was translated in a reticulocyte lysate and the efficiencies were compared with total liver and skeletal muscle RNA. The translational products of poly (A+) RNA were analyzed in a two dimensional gel. They show that many muscle specific proteins have been synthesized in vitro, which indicates that at least, part of the RNAs has remained intact.

Animals↗
PubMed1981

Hormonally stimulated adenylate cyclase and cAMP dependent protein kinase in membranes of rabbit erythroid cells separated according to density.

Plasma membranes were prepared after density gradient separation of erythroid cells obtained from bled animals. In a fraction enriched in young reticulocytes (lowest density), the basal and the prostaglandin stimulated adenylate cyclase were greatly augmented if compared with the membranes from unfractionated cells or from the layers of higher densities. Potentiation by GTP or soluble factor(s) of the prostaglandin stimulated adenylate cyclase was found solely in the fractions containing the youngest cells (lowest density). A very significant augmentation of both the basal and the effectors stimulated adenylate cyclase was obtained when the white blood cells and the platelets were removed by filtration through alpha-cellulose prior to density separation. A small population of probably very young reticulocytes was shown to contain a very active adenylate cyclase coupled to the hormonal receptor. Upon short time of maturation this coupling could no longer be detected. The cAMP generated in the fraction enriched in young reticulocytes increased the phosphorylation of some membrane proteins. The presence of a hormonally regulated adenylate cyclase and eventually the phosphorylation of some specific membrane proteins by the cAMP generated in situ permit to envisage possible functions of this system in young reticulocytes.

Adenylyl Cyclases↗
PubMed1980

A spin label study of the erythrocyte membranes in Duchenne muscular dystrophy.

Red blood cells and freshly prepared erythrocyte membranes of 15 patients with Duchenne muscular dystrophy (DMD) as well as age-matched controls were studied by the spin label method. No significant modifications appeared for spin-labelled proteins of ghost membranes. With the two fatty acid spin labels, 5-nitroxide stearate and 16-nitroxide stearate, we have confirmed previous results of Sato et al. concerning the thermal behaviour of the erythrocyte membranes, i.e. no change near the polar part probed by 5-nitroxide stearate and a linearization of the fluidity versus temperature variation around 12 degrees C, as explored by 16-nitroxide stearate. Furthermore we studied in the whole erythrocyte the amplitude of the 5-nitroxide stearate electron spin resonance signal as a function of the microwave power. This saturation effect was observed in 12 out of 15 controls and only in 1 out of 13 DMD cases studied. In erythrocyte membranes labelled with 16-nitroxide stearate the penetration of the label inside membranes was statistically different between DMD and controls. These new findings furnish further arguments in favour of a structural alteration of the phospholipid organization of erythrocyte membranes in DMD. Associated together, these different sets of tests obtained by spin labelling permit good statistical discrimination between DMD and normal subjects.

Adolescent↗
PubMed1980

Phosphorylation and dephosphorylation reactions by erythrocyte plasma membrane enzymes.

Human erythrocyte membranes contain a phosphoprotein phosphatase able to dephosphorylate membrane protein previously phosphorylated by the endogenous protein kinase. The level of dephosphorylation obtained after prolonged incubation is about one half of the phosphorylated residues. The characteristics of this enzyme are similar to those described for the cytoplasmic phosphoprotein phosphatase. In a membrane preparation the phosphorylation and dephosphorylation reactions can be repeated, at least twice, achieving similar levels of phosphate esterified or hydrolyzed. The coordination of these two enzyme systems might play a role in some of the functions attributed to the protein kinase system.

Erythrocyte Membrane↗